Cross-linking polypeptide that induces apoptosis

ABSTRACT

Described is a polypeptide comprising at least four domains specifically binding to a certain MHC peptide complex, the domains separated by linker amino acid sequences, thereby providing each domain with the capability to bind a separate MHC peptide complex, to a nucleic acid molecule encoding such a polypeptide, to a vector comprising such a nucleic acid molecule, to a host cell for expression of such a polypeptide, to a pharmaceutical composition comprising such a polypeptide, and to a kit of parts comprising at least two polypeptides of the disclosure.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 15/346,445, filed Nov. 8, 2016, pending, which is a continuation of U.S. patent application Ser. No. 13/976,952, filed Nov. 12, 2013, now U.S. Pat. No. 9,512,231, issued Dec. 6, 2016, which is a national phase entry under 35 U.S.C. § 371 of International Patent Application PCT/NL2011/050893, filed Dec. 22, 2011, designating the United States of America and published in English as International Patent Publication WO2012/091564 A2 on Jul. 5, 2012, which claims the benefit under Article 8 of the Patent Cooperation Treaty to U.S. Application Ser. No. 61/460,213, filed Dec. 27, 2010 and Application Ser. No. 61/572,318, filed Jul. 13, 2011, the disclosure of each of which is hereby incorporated herein in its entirety by this reference.

TECHNICAL FIELD

The application relates to the field of medicine and biotherapeutics. It also relates to the field of tumor biology. More in particular, the application relates to specific binding molecules that induce apoptosis in tumor cells. More specifically, single-chain multivalent, preferably tetravalent or bigger, specifically hexavalent repeats of human antibody variable (heavy) fragments are provided that apparently cross-link MHC-peptide complexes on cells thereby inducing cell death. The application also relates to the use of these binding molecules in selectively killing cancer cells and other aberrant cells.

BACKGROUND

Since the sixties of the last century, it has been proposed to use the specific binding power of the immune system (T-cells and antibodies) to selectively kill tumor cells but leave alone the normal cells in a patient's body. Many tumor antigens that could be targeted by in particular antibodies, like carcino-embryonic antigen (CEA), alpha-fetoprotein (AFP) and so on have been suggested since those days, but for essentially all of these antigens expression is associated with normal tissue as well. Thus, so far this has been an elusive goal.

In an earlier application WO2007/073147 (Apoptosis-inducing protein complexes and therapeutic use thereof, incorporated herein by reference) disclosed is a polypeptide complex comprising at least six polypeptides in which polypeptides were assembled to form the complex via post-translational covalent or non-covalent non-peptide bond based linker chemistry. Although such a complex achieves the goal of (specifically) killing, e.g., tumor cells by inducing apoptosis in these tumor cells (although not wishing to be bound by theory, at present it is believed that this is the result of cross-linking), it is quite difficult to produce, since it requires post-translational assembly of polypeptides in functional complexes after expression thereof. In addition the stability of such a complex in vivo may be an issue of concern.

DISCLOSURE

Disclosed herein is killing aberrant (e.g., tumor) cells by apoptosis that can be achieved by providing a single-chain polypeptide comprising at least four domains specifically binding to a certain major histocompatibility complex (MHC)-peptide complex, the domains preferably separated by linker amino acid sequences of which the peptide backbone is incorporated in the peptide backbone of the polypeptide, thereby providing each domain with the capability to bind a separate MHC-peptide complex. More specifically, the disclosure relates to multiple recombinant antibody-fragments assembled at the DNA level into a single chain, which specifically bind MHC-peptide complexes and are able to induce cell-death, in particular apoptosis upon cross-linking of multiple MHC-peptide complexes. In particular, the application relates to methods of diagnosing and treatment of cancer using these recombinant multivalent single-chain polypeptides.

INTRODUCTION

The primary immunological function of MHC molecules is to bind and “present” antigenic peptides to form an MHC-peptide (MHC-p) complex on the surface of cells for recognition and binding by antigen-specific T-cell receptors (TCRs) of lymphocytes. With regard to their function, two classes of MHC-peptide complexes can be distinguished:

(i) MHC class I-peptide complexes can be expressed by almost all nucleated cells in order to attract CD8⁺ cytotoxic T-cells, and

(ii) MHC class II peptide complexes are constitutively expressed only on so-called antigen presenting cells (APCs), such as B-lymphocytes, macrophages or dendritic cells (DCs).

MHC class I-peptide complexes are composed of a variable heavy chain, invariable β-microglobulin and antigenic peptide. The MHC class II molecules are characterized by distinctive α and β polypeptide subunits that combine to form αβ heterodimers characteristic of mature MHC class II molecules. Differential structural properties of MHC-class I and -class II molecules account for their respective roles in activating different populations of T-lymphocytes. Cytotoxic T_(C) lymphocytes (CTLs) bind antigenic peptides presented by MHC class I molecules. Helper T_(H) lymphocytes bind antigenic peptides presented by MHC class II molecules. MHC class I and class II molecules differentially bind CD8 and CD4 cell adhesion molecules. MHC class I molecules are specifically bound by CD8 molecules expressed on cytotoxic T_(C) lymphocytes, whereas MHC class II molecules are specifically bound by CD4 molecules expressed on helper T_(H) lymphocytes.

The sizes of the antigenic peptide-binding pockets of MHC class I and class II molecules differ; class I molecules bind smaller antigenic peptides, typically 8-10 amino acid residues in length, whereas class II molecules bind larger antigenic peptides, typically 13-18 amino acid residues in length.

In humans, MHC molecules are termed human leukocyte antigens (HLA). HLA-associated peptides are short, encompassing typically 9-25 amino acids. Humans synthesize three different types of class I molecules designated HLA-A, HLA-B, and HLA-C. Human class II molecules are designated HLA-D, e.g., HLA-DR.

The MHC expressed on all nucleated cells of humans and of animal cells plays a crucial role in immunological defense against pathogens and cancer. The transformation of normal cells to aberrant cancer cells involves several major changes in gene expression. This results in profound changes in the antigenic composition of cells. It is well established that new antigenic entities are presented as MHC-restricted tumor associated antigens. As such, the MHC class I and class II system may be seen as nature's proteomic scanning chip, continuously processing intracellular proteins, generating antigenic peptides for presentation on the cell surface. If these antigenic peptides elicit an immune reactivity the transformed cells are killed by the cellular immune system. However, if the transformed cells resist immune mediated cell killing, cancer may develop.

Antibodies that bind MHC class I molecules on various cell types have been studied in detail for their mode of action. Mouse monoclonal antibodies, that bind the MHC class I α1 domain of the MHC class I α chain induce apoptosis in activated T-cells, but not in resting T-cells. Other reports mention antibodies specific for, e.g., the α3 domain of MHC class I, which induce growth inhibition and apoptosis in B-cell derived cancer cells. However, in this case a secondary, cross-linking antibody was required for the induction of apoptosis (A. E. Pedersen et al., Exp. Cell Res. 1999, 251:128-34).

Antibodies binding to β2-microglobulin (β2-M), an essential component of the MHC class I molecules, also induce apoptosis. Several hematologic cancer cells treated with anti-β2M molecules were killed efficiently, both in vitro and in vivo (Y. Cao et al., Br. J. Haematol. 2011, 154:111-121).

Thus, it is known that binding of MHC class I or MHC class II molecules by several anti-MHC antibodies can have an apoptosis-inducing effect. However, the therapeutic application of the currently available anti-MHC antibodies has been hampered by the lack of target cell specificity. Since known antibodies are directed primarily against an epitope of the MHC molecule itself (e.g., HLA-DR), the cell surface expression of the MHC epitope determines whether or not a cell can be triggered to undergo apoptosis. Because MHC class I and MHC class II molecules are expressed on both normal and diseased cells, it is clear that these antibodies cannot discriminate between normal and abnormal (e.g., tumor and/or aberrant) cells. As a consequence, their therapeutic value is significantly reduced if not abolished by the side-effects caused by unwanted apoptosis of healthy cells. Antibodies that specifically recognize MHC-presented peptides derived from cancer antigens, on the surface of aberrant cells would therefore dramatically expand the therapeutic repertoire, if they could be shown to have anti-cancer cell activity, leading to the eradication of cancer. In addition, methods hereof to induce apoptosis via MHC-class I or MHC class II may depend on external cross-linking of anti-MHC antibodies.

Obtaining antibodies binding to MHC-peptide complexes remains a laborious task and several failures have been reported. The first available antibodies have been obtained after immunization of mice with recombinant MHC-peptide complexes or peptide-loaded TAP-deficient antigen presenting cells, and more recently by selection from phage-antibody libraries made from immunized transgenic mice or by selection from completely human antibody phage libraries. Immunization with MHC-peptide complexes is extremely time-consuming. Moreover, antibodies of murine origin cannot be used repetitively in patients because of the likely development of a human anti-mouse antibody response (so-called anti-drug antibodies, ADA). Antibodies derived from phage display in general display low affinity for the antigen and thus may require additional modifications before they can be used efficiently. The antibody specificities are preferably selected through phage (or yeast) display, whereby an MHC molecule loaded with a cancer related peptide is presented to the library. Details are given in the experimental part. It is also possible to employ (transgenic) mice to obtain domains specifically recognizing the MHC-peptide complex. It has been reported that a single chain MHC-peptide molecule can be produced mimicking the peptide MHC complex. E.g., mice having part of a human immune system can be immunized with such a single chain molecule. The antibody specificities hereof may be checked for specificity to the MHC-peptide complex and should not recognize (to any significant extent) empty MHC (although this is less relevant since at least empty MHC-1 is not stable) or MHC loaded with irrelevant peptides or the peptides by themselves.

Disclosed is at least partially overcoming the above listed limitations and providing a pharmaceutically active molecule that specifically and efficiently induces cell death, in particular apoptosis and that at the same time is manufactured in a less cumbersome manner, i.e., as a multivalent single-chain protein. In particular, disclosed is a method to specifically and selectively induce apoptosis of cells of interest, for example, of aberrant cells like tumor cells and/or autoimmune disease related aberrant cells expressing a tumor antigen, leaving healthy cells essentially unaffected. MHC-1 peptide complexes are a valuable target for tumors of almost any origin, whereas MHC-2 peptide complexes are valuable targets for tumors of hematopoietic origin. In addition to tumors, MAGE expression has also been shown in cells involved in Rheumatoid Arthritis (D. K. McCurdy et al., J. Rheumatol. 2002, 29:2219-2224).

Provided is a polypeptide comprising at least four domains specifically binding to a certain MHC-peptide complex, the domains separated by linker amino acid sequences, thereby providing each domain with the capability to bind a separate MHC-peptide complex. Typically, a single polypeptide comprising all necessary MHC-peptide complex-binding domains separated by amino acid sequences is provided. This does not mean that every molecule hereof may only consist of a single polypeptide chain binding to MHC-peptide alone. It is, e.g., possible to provide other binding domains with non-MHC-peptide specificity on the single chain polypeptide comprising the MHC-peptide complex binding domains. The second binding domain would typically not comprise antibody-derived binding domains like the first domains, but would be a domain conferring other desirable properties on the binding polypeptide, such as, but not limited to, improved half-life. As an example, the addition of Human Serum Albumin (HSA) on the binding polypeptide may be useful for extension of half-life, etc. The molecules hereof may also comprise a binding domain for molecules, such as HSA, so that HSA may be bound afterwards.

Although not wishing to be bound to theory, it does seem that the MHC-peptide complex binding domains result in a close co-localization (referred to herein as cross-linking) of several MHC-1 molecules (in the present specification most of the time MHC-1 will be mentioned. The disclosure is equally applicable with MHC-2) on the cell membrane, which in turn leads to cell death. The number of MHC-1 molecules that need to be co-localized may vary, but consistent results have been seen with four MHC-peptide complex binding domains in the binding molecule and upward.

The MHC-peptide complex binding domains on the polypeptide may be identical or different, but for specificity's sake, most of them must recognize the complex of MHC-1 loaded with a relevant peptide. The requirement is a functional one. The polypeptides hereof must be able to cross-link MHC-1 loaded molecules on tumor cells, but should not cross-link MHC-1 molecules loaded with a different non-tumor associated peptide or MHC-1 on a normal cell to any significant extent. It is, therefore, preferred that all MHC-peptide complex binding domains recognize the same MHC-1-peptide complex (and essentially only in tumor associated peptide loaded form). For ease of selection and production, the MHC-peptide complex binding domains are preferably identical. If they are not identical, they preferably recognize the same epitope, or at least the same MHC-1-peptide complex. A binding domain must at least be capable of specifically binding to the MHC-1-peptide complex with sufficient affinity to result in binding to essentially only the MHC-1 peptide complexes they were developed against. Many MHC-peptide complex binding domains are well known to people of skill in the art. Immediately apparent are MHC-peptide complex binding domains derived from the immune system, such as single chain T-cell receptor domains and immunoglobulin domains and fragments of immunoglobulins. Preferably, the domains and fragments are 100 to 150 amino acids long. Preferably, the MHC-peptide complex binding domains are similar to variable heavy domains or light domains (Vh or VI) of antibodies. A good source for such MHC-peptide complex-binding domains are phage display libraries. In another embodiment, at least one of the specific binding domains comprises a single chain T-cell receptor domain.

Throughout the specification, the term “fragment” refers to an amino acid sequence, which is part of a protein domain or which builds up an intact protein domain. Fragments hereof have binding specificity for the respective target.

The techniques of connecting proteinaceous domains in a single molecule are many and well known. Whether the MHC-peptide complex binding domains, from now on also referred to as “binding domains” throughout the specification, are actually selected from a library physically or whether only the information (sequence) is only used is of little relevance.

The binding domains on the polypeptide are typically separated by a linker amino acid sequence, although binding domains in which some amino acids on the boundaries are not involved in binding the target are present (flanking sequences) may not require linkers. The linkers between the binding domains may be the same or different. In many instances, simple Gly-Ser linkers of 4-15 amino acids may suffice, but if greater flexibility of the amino acid chain is desired longer or more complex linkers may be used. Preferred linkers are (Gly₄Ser)_(n)(SEQ ID NO:18), (GSTSGS)_(n) (SEQ ID NO:19), GSTSGSGKPGSGEGSTKG (SEQ ID NO:20), EFAKTTAPSVYPLAPVLESSGSG (SEQ ID NO:21) or any other linker that provides flexibility for protein folding and stability against protease. Another group of preferred linkers are linkers based on hinge regions of immunoglobulins. These linkers tend to be quite flexible and quite resistant to proteases. Examples are given in the experimental part. The most preferred linkers are EPKSCDKTHT (IgG1) (SEQ ID NO:22), ELKTPLGDTTHT (IgG3) (SEQ ID NO:23), and ESKYGPP (IgG4) (SEQ ID NO:24). The binding domains may be separated only by a linker, but other useful amino acid sequences may be introduced between the binding domains or at the N-terminus or at the C-terminus of the first or last binding domain sequence, respectively. Thus, in one embodiment, provided is a polypeptide as given above, further comprising an amino acid sequence having an additional function, preferably an effector function. Although one of the advantages of the disclosure is ease of production and the simplicity of the molecules hereof, the choice for a single nucleic acid encoding all necessary functions in itself enables the relatively easy addition (to the extent that there is room in the chosen expression vectors, etc.) of other functionalities in the resulting polypeptide. The possibilities are many. It is possible to introduce an effector molecule, e.g., a payload, such as a toxin or an apoptosis inducing molecule. It is at present not known how many cross-linked MHC-1 peptide complexes are necessary per cell to induce apoptosis. If only one cross-linked complex would suffice then a payload may be not really be useful. If more than one cross-linked complex is necessary then a payload may be helpful in those cases where the cell has been reached by the molecule, but not enough cross-linked complexes are formed. In that case, if and when the cross-linked complex is internalized (as is expected) then the payload can have its (cytotoxic) function. It is preferred that such a payload has a contribution to the specificity of the cytotoxic effect. Therefore, it is preferred to use as a payload a polypeptide that induces cell death in aberrant cells, but not in normal cells. Such a polypeptide is apoptin or a number of its fragments and/or derivatives. Other examples of cytotoxic polypeptides include, but are not limited to, cholera toxin, ricin A, etc., other functions that may be introduced may have to do with improved half-life (HSA can be included) or complement activation (Fc part of immunoglobulins, in this case the molecules hereof may dimerize). Other functionalities that can be incorporated are cytokines, hormones, Toll-like receptor ligands, etc.

The number of binding domains necessary to provide sufficient cross-linking will undoubtedly vary with the tumor that it is targeted. Different tumors will have different levels of MHC-1/MHC-2 expression, different levels of peptide presentation, etc. It is expected that 4-12 binding domains per polypeptide chain will be optimal. There is however no real upper limit, except for tissue penetration, expression and production issues. For ease of production, hexamers (which have shown excellent results in animal models) are preferred. Therefore, provided is a polypeptide having six MHC-peptide complex binding domains.

As stated before, the binding domains are preferably based on, or derived from immunoglobulin domains or fragments of domains (or comparable single chain T-cell receptor domains or other binding proteins). The immunoglobulin domains should have at least one CDR-like domain or one domain comprising one or more CDR-like loops, preferably, however, three domains. These CDR-like domains should be separated by (framework) domains that present the CDR-like regions in a proper manner. A suitable domain is a Vh domain of a human antibody. This domain may be “camelized,” meaning that a number of amino acid residues have been replaced by amino acid residues from camelids, such as in the llama Vh. Preferred substitutions are E6A, A33C, V37F, G44E, L45R, W47G, S74A, R83K, A84P or L108Q. Thus, provided is a polypeptide, wherein at least one, but preferably all of the specific binding domains comprise an immunoglobulin fragment. The origin or the method of selection as well as the method of production of the immunoglobulin fragments to be used in the polypeptides, according to the disclosure is not really relevant. According to one embodiment, a polypeptide comprises at least one, preferably more than one, immunoglobulin fragment that is a natural, mutated and/or synthetic VH.

Although many different combinations of MHC and peptides are contemplated, the most preferred is the combination of MHC-1 and a peptide from a tumor related antigen presented by the MHC-1. Because of HLA restrictions, there are many combinations of MHC-1 peptide complexes as well as MHC-2 peptide complexes that can be designed based on the rules for presentation of peptides in MHC. These rules include size limits on peptides that can be presented in the context of MHC, restriction sites that need to be present for processing of the antigen in the cell, anchor sites that need to be present on the peptide to be presented, etc. The exact rules differ for the different HLA classes and for the different MHC classes. It is found that MAGE peptides are very suitable for presentation in an MHC context. An MHC-1 presentable peptide with the sequence Y-L-E-Y-R-Q-V-P-G (SEQ ID NO:7) in MAGE-A was identified, that is present in almost every MAGE-A variant; MAGE-A1-MAGE-A12, and that will be presented by one of the most prevalent MHC-1 alleles in the Caucasian population (namely, HLA-A0201). A second MAGE peptide that is presented by another MHC-1 allele (namely, HLA-CW7) and that is present in many MAGE variants, like, for example, MAGE-A2, -A3, -A6 and -A12, is E-G-D-C-A-P-E-E-K (SEQ ID NO:8). These two combinations of MHC-1 and MAGE peptides together would cover 80% of the Caucasian population. It is shown, in vitro, that tumor cell lines with the correct HLA alleles present are efficiently killed by the molecules. The same approach can be followed for other MHC molecules, other HLA restrictions and other tumor-associated antigens. Relevant is that the chosen peptide to elicit the response must be presented in the context of an MHC molecule and recognized in that context only. Furthermore, the peptide must be derived from a sufficiently tumor-specific antigen and the HLA restriction must occur in a relevant part of the population. One of the important advantages of the disclosure is that tumors that down-regulate their targeted MHC-peptide complex, can be treated with a second binding molecule against a different MHC-peptide complex based on the same antigen. If this one is down-regulated a third one will be available. For heterozygotes, six different targets on MHC may be available. Since cells need to be “inspected” by the immune system from time to time, escape through down-regulation of all MHC molecules does not seem a viable escape route. In the case that MAGE is the antigen from which the peptide is derived escape through down-regulation of the antigen is also not possible, because MAGE seems important for survival of the tumor (L. Marcar et al., Cancer Res. 2010, 70:10362-10370). Thus, the disclosure, in an important aspect reduces or even prevents escape of the tumor from the therapy, in the sense that the tumor remains treatable.

Because MHC molecules are used as a target, and individuals differ in the availability of MHC targets, also provided is a so-called companion diagnostic to determine the HLA composition of an individual. Although the disclosure preferably uses a more or less universal (MAGE) peptide, it also provides a diagnostic for determining the expression of the particular antigen by the tumor. In this manner, the therapy can be geared to the patient, particularly also in the set-up to prevent escape, as described herein before. It is known that the HLA restriction patterns of the Asian population and the black population are different than that of the Caucasian population. For these populations, different MHC-peptide complexes can be targeted, as described in the detailed description.

Although the disclosure presents more specific disclosure on tumors, it must be understood that other aberrant cells can also be targeted by the molecules of the disclosure. These other aberrant cells are typically cells that also proliferate without sufficient control. This occurs in autoimmune diseases. It is typical that these cells start to show expression of tumor antigens. In particular, MAGE polypeptides have been identified in Rheumatoid Arthritis. Thus, provided in a preferred embodiment is a polypeptide hereof, whereby the specific binding domains are capable of binding to an MHC-I-peptide complex. In a further preferred embodiment, also provided is a polypeptide whereby the specific binding domains are capable of binding to MHC-I-peptide complexes comprising a peptide derived from a tumor related antigen, in particular MHC-I-peptide complexes comprising a variety of MAGE peptides.

An “aberrant cell” is defined as a cell that deviates from its usual and healthy normal counterparts and shows uncontrolled growth characteristics.

One of the polypeptides, exemplified herein, has binding domains with the amino acid sequence essentially corresponding to:

(SEQ ID NO: 11, AH5) QLQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGVAV ISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGS YYVPDYWGQGTLVTVSS.

Another one has binding domains comprising the amino acid sequence:

(SEQ ID NO: 12, 11H) EVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWLSY ISSDGSTIYYADSVKGRFTVSRDNAKNSLSLQMNSLRADDTAVYYCAVSP RGYYYYGLDLWGQGTTVTVSS.

One polypeptide has an amino acid sequence essentially corresponding to:

(SEQ ID NO: 4, Hexa-AH5) MAQLQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGV AVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAG GSYYVPDYWGQGTLVTVSSGSTSGSMAQLQLQESGGGVVQPGRSLRLSCA ASGFTFSSYGMHWVRQAPGKEREGVAVISYDGSNKYYADSVKGRFTISRD NSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPDYWGQGTLVTVSSGSTSGS MAQLQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGV AVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAG GSYYVPDYWGQGTLVTVSSGSTSGSMAQLQLQESGGGVVQPGRSLRLSCA ASGFTFSSYGMHWVRQAPGKEREGVAVISYDGSNKYYADSVKGRFTISRD NSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPDYWGQGTLVTVSSGSTSGS MAQLQLQESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGV AVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAG GSYYVPDYWGQGTLVTVSSGSTSGSMAQLQLQESGGGVVQPGRSLRLSCA ASGFTFSSYGMHWVRQAPGKEREGVAVISYDGSNKYYADSVKGRFTISRD NSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPDYWGQGTLVTVSS. Or: (SEQ ID NO: 13, Hexa-11HCH1) EVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGL EWLSYISSDGSTIYYADSVKGRFTVSRDNAKNSLSLQMNSLRADDTAVYY CAVSPRGYYYYGLDLWGQGTTVTVSSEPKSCDKTHTAEVQLVQSGGGLVK PGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWLSYISSDGSTIYYADS VKGRFTVSRDNAKNSLSLQMNSLRADDTAVYYCAVSPRGYYYYGLDLWGQ GTTVTVSSEPKSCDKTHTAEVQLVQSGGGLVKPGGSLRLSCAASGFTFSD YYMSWIRQAPGKGLEWLSYISSDGSTIYYADSVKGRFTVSRDNAKNSLSL QMNSLRADDTAVYYCAVSPRGYYYYGLDLWGQGTTVTVSSEPKSCDKTHT AEVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWLS YISSDGSTIYYADSVKGRFTVSRDNAKNSLSLQMNSLRADDTAVYYCAVS PRGYYYYGLDLWGQGTTVTVSSEPKSCDKTHTAEVQLVQSGGGLVKPGGS LRLSCAASGFTFSDYYMSWIRQAPGKGLEWLSYISSDGSTIYYADSVKGR FTVSRDNAKNSLSLQMNSLRADDTAVYYCAVSPRGYYYYGLDLWGQGTTV TVSSEPKSCDKTHTAEVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMS WIRQAPGKGLEWLSYISSDGSTIYYADSVKGRFTVSRDNAKNSLSLQMNS LRADDTAVYYCAVSPRGYYYYGLDLWGQGTTVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC. Or: (SEQ ID NO: 17, Hexa-11HAH5) EVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLE WLSYISSDGSTIYYADSVKGRFTVSRDNAKNSLSLQMNSLRADDTAVYYC AVSPRGYYYYGLDLWGQGTTVTVSSGGGGSGGGGSGGGSQLQLQESGGGV VQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGVAVISYDGSNKYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPDYWGQGT LVTVSSGSTSGSGKSPGSGEGTKGEVQLVQSGGGLVKPGGSLRLSCAASG FTFSDYYMSWIRQAPGKGLEWLSYISSDGSTIYYADSVKGRFTVSRDNAK NSLSLQMNSLRADDTAVYYCAVSPRGYYYYGLDLWGQGTTVTVSSEFAKT TAPSVYPLAPVLESSGSGQLQLQESGGGVVQPGRSLRLSCAASGFTFSSY GMHWVRQAPGKEREGVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAGGSYYVPDYWGQGTLVTVSSGGGGSGGGGSGGGGS EVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWLSY ISSDGSTIYYADSVKGRFTVSRDNAKNSLSLQMNSLRADDTAVYYCAVSP RGYYYYGLDLWGQGTTVTVSSGSTSGSGKSPGSGEGTKGQLQLQESGGGV VQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKEREGVAVISYDGSNKYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGSYYVPDYWGQGT LVTVSS.

The disclosure, of course, comprises the polynucleotides encoding the polypeptides. The molecules can be produced in prokaryotes as well as eukaryotes. The codon usage of prokaryotes may be different from that in eukaryotes. The nucleic acids can be adapted in these respects. Also, elements that are necessary for secretion may be added, as well as promoters, terminators, enhancers, etc. In addition, elements that are beneficial or necessary for isolation and/or purification may be added. Typically, the polynucleotides are provided in an expression vector suitable for the host in which they are to be produced. Choice of a production platform will depend on the size of the molecule, the expected issues around folding, whether additional sequences are present that require glycosylation, etc., thus, typically, nucleic acids are adapted to the production platform in which the polypeptides are to be produced. Thus, provided is a polynucleotide encoding a polypeptide as well as an expression vector comprising such a polynucleotide. For stable expression in a eukaryote, it is preferred that the polynucleotide encoding the polypeptide is integrated into the host cell genome (at a suitable site that is not silenced). Thus, the disclosure comprises in a particular embodiment: a vector comprising means for integrating the polynucleotide in the genome of a host cell.

Further described is the host cell or the organism in which the polypeptide encoding nucleic acid molecule is present and which is capable of producing the polypeptide.

Included in the disclosure are also the methods for producing a polypeptide comprising culturing a host cell comprising a nucleic acid molecule allowing for expression of the nucleic acid and harvesting a polypeptide.

For administration to subjects, the polypeptides are formulated. Typically, these polypeptides will be given parenterally. For formulation, simply water (saline) for injection may suffice. For stability reasons more complex formulations may be necessary. Also contemplated are lyophilized compositions as well as liquid compositions, provided with the usual additives. Thus, provided is a pharmaceutical composition comprising a polypeptide and suitable diluents and/or excipients.

The dosage of the polypeptides is established through animal studies and clinical studies in so-called rising-dose experiments. Animal experiments so far have not shown any relevant toxicity at effective dosages. Typically, the doses will be comparable with present day antibody dosages (at the molar level, the weight of the invented molecules may differ from that of antibodies). Typically, such dosages are 3-15 mg/kg body weight, or 25-1000 mg per dose.

It has been established in the field of tumor therapy that a single agent is hardly ever capable of eradication of tumor from a patient. Especially in the more difficult to treat tumors, the first applications of the polypeptides will (at least initially) probably take place in combination with other treatments (standard care). Thus, also provided is a pharmaceutical composition comprising a polypeptide and a conventional cytostatic and/or tumoricidal agent. Moreover, also provided is a pharmaceutical composition comprising a polypeptide for use in an adjuvant treatment of cancer. Additionally, also provided is a pharmaceutical composition comprising a polypeptide for use in a combination chemotherapy treatment of cancer.

The pharmaceutical compositions hereof will typically find their use in the treatment of cancer, particularly in forms of cancer where the targets of the preferred single-chain polypeptides (complexes of MHC and MAGE-A peptides) are presented by the tumors. Table 1 gives a list of tumors on which these targets have been found. It is easy using a binding domain hereof to identify tumors that present the target MHC-peptide complexes. This can be done in vitro or in vivo (imaging).

The terms “repeat” and “repeats” have the same meaning as “domain” and “domains,” respectively, throughout the specification. The term “binding” is defined as interactions between molecules that can be distinguished from background interactions. The term “specific,” for example, in “specific binding (domain),” has the meaning of indicating a molecule that can have an interaction with another molecule with higher binding affinity than background interactions between molecules. Typically, the polypeptides hereof do not need high affinity binding domains, since they benefit from the so-called avidity effect. Similarly, the term “specificity” refers to an interaction, for example, between two molecules or between a cell and a molecule that has higher binding affinity than background interactions between molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Specific binding of HLA-A0201/multi-MAGE-A-specific phage clones isolated from a large human non-immune antibody Fab phage library. Individual antibody Fab expressing phages that were selected against biotinylated HLA-A0201/multi-MAGE-A were analyzed by ELISA for their capacity to bind the relevant peptide/MHC complex only. Streptavidin coated 96-well plates were incubated with soluble HLA-A0201/multi-MAGE-A (A2/multiMage) or HLA-A0201/JCV (A2/JC) peptide/MHC complexes (10 μg/ml), washed to remove non-bound complexes and incubated with individual phage clones. Non-binding phages were first removed by three washes with PBS/TWEEN®, followed by incubation with anti-M13 antibody (1 μg/ml, Amersham) for one hour by room temperature. Finally, the wells were incubated with an HRP-labeled secondary antibody and bound phages detected.

FIG. 2: Phages AH5, CB1 and CG1 specifically bind cells presenting the multi-MAGE-A peptide. Phages AH5, CB1, CG1, BD5 and BC7 that had shown specific binding in ELISA using the relevant HLA-A201/multi-MAGE-A complex and an irrelevant HLA-A201 complex loaded with a JCV peptide were analyzed for their capacity to bind cells presenting the multi-MAGE-A peptide in HLA-A0201 molecules. To this end, human B-LCL (BSM) were loaded with multi-MAGE-A peptide (10 μg in 100 jil PBS) for 30 minutes at 37° C., followed by incubation with the Fab phages AH5, CB1, CG1, BD5 and BC7 and analyzed by flow-cytometry using anti-phage antibodies and a fluorescently labeled secondary antibody.

FIG. 3: Phages expressing HLA-A2/multi-MAGE-A-specific Fab bind tumor cells of distinct histologic origin. Phages AH5, CB1 and CG1 specific for HLA-A0201/multi-MAGE-A and a positive control phage specific for HA-0101/MAGE-A1 were used for staining of distinct tumor cell lines. To this end the prostate cancer cell line LNCaP, the multiple myeloma cell line MDN, the melanoma cell lines MZ2-MEL43 and G43, and the breast cancer cell line MDA-MD157 were incubated with the different phages (30 minutes at 4° C.), bound phages were then detected by flow cytometry using anti-phage antibodies and fluorescently labeled secondary antibodies.

FIG. 4: Phage AH5 specifically binds HLA-A0201/multiMAGE-A complexes only. To determine specificity of the phage AH5 an ELISA was performed using relevant and irrelevant peptide/MHC complexes. HLA-A0201 with multi-MAGE-A, gp100, JCV and MAGE-C2 peptides, as well as HLA-A1 with MAGE-A1 peptide were coated on streptavidin 96-well plates and incubated with phage AH5.

FIG. 5: Hexa-AH5 is expressed by bacteria. Expression of the Hexa-AH5 gene in pStaby 1.2 was induced after addition of IPTG to SE-1 bacteria. Bacteria were grown in TYAG medium at 30° C. until OD600=0.8. At that time, medium was replaced with TY medium supplemented with IPTG, and bacteria allowed to grow for four hours. Medium and periplasm were collected and analyzed by 10% SDS-PAGE.

FIG. 6: Microscopic analysis of Hexa-AH5-treated Daju cells reveals apoptosis. Daju cells cultured in DMEM medium supplemented with pen/strep, glutamine and non-essential amino acids were treated with heza-AH5 protein (10 μg/ml total) for four hours and inspected by microscopy for signs of apoptosis.

FIG. 7: Treatment with Hexa-AH5 induces active caspase-3. Daju cells were treated with 10 μg/ml Hexa-AH5 protein for four hours. Next, a caspase-3 inhibitor was added (FAM-DEVD-FMK) and 1 hour later cells were analyzed by fluorescence microscopy.

FIG. 8: Intratumoral injection of Hexa-AH5 induces apoptosis in a transplantable human tumor model. The human prostate tumor cell line PC346C was injected into the prostate of NOD-Scid mice and allowed to grow until visible by ultrasound inspection. The mice then received an intratumoral injection of Hexa-AH5 (10 μg total in 20 μl volume) or PBS as control. The next day, the mice were sacrificed, tumors removed and paraffin embedded. Tumor slices were stained for fragmented DNA and analyzed by microscopy. Results show large areas of apoptotic cells (stained dark) only after treatment with Hexa-AH5. No signs of apoptosis were detected in PBS-treated mice.

FIG. 9: Intravenous injection of Hexa-AH5 results in apoptotic prostate tumor cells in the orthotopic mouse tumor model. NOD-scid mice with orthotopic PC346C prostate tumor were injected once with 25 μg Hexa-AH5 (in 100 μl total volume). The next day, mice were sacrificed and tumors removed. Paraffin-embedded tumor slices were stained for fragmented DNA and analyzed by microscopy. Results show large areas of apoptotic cells in treated mice only.

FIG. 10: Intravenous treatment with Hexa-AH5 of mice with orthotopic prostate cancer results in activation of caspases. NOD-scid mice with orthotopic PC346C prostate tumor were injected once with 25 μg Hexa-AH5 (in 100 μl total volume). The next day, mice received an intravenous injection with a universal caspase inhibitor (FLIVO), which was allowed to circulate for one hour. Mice were then sacrificed and tumors removed. Paraffin-embedded tumor slices were analyzed by fluorescence microscopy, which revealed active caspase in Hexa-AH5-treated mice only.

FIGS. 11A and 11B: Treatment with Hexa AH5-Fc and AH5-HSA induces active caspase-3. Melanoma 624 cells incubated for 24 hours with supernatant obtained from 293T cells transfected with the pcDNA-3.1/Hexa AH5-Fc (FIG. 11A) or/Hexa AH5-HSA (FIG. 11B) constructs demonstrate presence of active caspase-3. Active caspase-3 in melanoma 624 cells was detected by fluorescence microscopy 4 hours after incubation with FAM-DEVD-FMK (SEQ ID NO:25).

FIG. 12: mouse survival and tumor growth after i.v. treatment with hexameric AH5 protein. Melanoma Daju cells were subcutaneously injected into NOD-SCID mice. When palpable tumors were present, mice were intravenously injected with hexameric AH5 (2.5 μg/2 times/week). Tumor growth and survival was determined.

FIG. 13: schematic presentation of possible hexameric proteins. Hexameric proteins may be composed of distinct building blocks, such as: 1) distinct linker sequences and 2) distinct V_(H) domains. Shown are a number of possible combinations.

FIGS. 14A and 14B: Expression of Hexameric AH5 at 25° C. SE-1 bacteria containing the Hexameric AH5 construct were grown and induced at 25° C. FIG. 14A, instant blue staining of SDS-PAGE gel: lane 1-periplasm of induced SE-1 pStaby 1.2-Hexa-AH5, lane 2: protein marker (M). FIG. 14B, western blot with anti-cMyc antibody: lane 1: Hexa-AH5, lane 2 protein marker (M).

DETAILED DESCRIPTION

As outlined in previous international patent application publication WO2007/073147, the desired specific and selective killing of aberrant cells via the apoptosis machinery can be achieved by contacting these cells with a multivalent mono-specific protein complex comprising multiple antigen-specific MHC-restricted single chain T-cell receptors (TCRs) and/or MHC-restricted antigen-specific antibodies, which antigen is expressed by the targeted aberrant cells and presented in the context of MHC molecules. This finding then, opened the possibility to selectively kill a population of cells that are positive for a certain MHC-peptide complex of interest, for example, tumor cells expressing HLA class I molecules in complex with peptides derived from tumor-associated antigens.

Without wishing to be bound by theory, and based on in the disclosure in this application, it is thought that a multivalent like, for example, a hexavalent mono-specific protein induces apoptosis via the clustering of a number of (identical) MHC-p complexes on the cell surface of a target cell. The data shown in the previous application WO2007/073147 suggest that clustering of three MHC-p complexes may not be sufficient for apoptosis induction, whereas a hexavalent complex is very efficient in inducing apoptosis. Thus, it is disclosed now that apoptosis induction requires the binding of at least four, preferably at least five, more preferably at least six MHC-p complexes by one multivalent single-chain protein.

The terms “protein” and “polypeptide” have roughly the same meaning throughout the text of this application and refer to a linear proteinaceous sequence comprising two or more linked amino acid residues. In the context of the proteins and protein complexes that specifically bind to MHC-p complexes, “binding molecules” and “polypeptides” have the same meaning as “protein” and “protein complexes.” The term “apoptosis” refers to the process of programmed cell death.

In one embodiment, a multivalent single-chain protein encompasses four, five, six, seven, eight, nine, ten, eleven or twelve domains or clusters of domains, each domain or cluster of domains capable of recognizing and binding to a specific MHC-peptide complex. In contrast to the known methods for apoptosis induction using anti-MHC antibodies, a multivalent single-chain monomeric protein, disclosed herein, can induce apoptosis itself and does not require any external post-translational cross-linking. The multiple domains or multiple clusters of domains are connected to form a linear sequence at the DNA level and thus connected into a linear single-chain monomeric polypeptide via regular peptide bonds at the protein level.

Disclosed is a multivalent single-chain protein comprising at least four and preferably six domains or clusters of domains capable of recognizing and binding to a specific MHC-peptide complex. At least four or preferably six domains or clusters of domains preferably recognize the same MHC-peptide complex, i.e., the preferred multivalent single-chain protein is mono-specific with respect to the MHC-p complex. The domains of the multivalent single-chain protein that specifically recognize and bind to a MHC-p complex can be TCR domains or a functional fragment thereof (together herein referred to as TCRs) and/or an antibody that mimics TCR specificity, for example, a genetically engineered antibody, such as a single-chain variable fragment (scFv) or the variable domain V of the heavy chain H of an antibody (referred to throughout the text as VH, Vh or V_(H)). Also, a multivalent single-chain protein hereof may encompass TCR domains as well as MHC class-restricted antibody domains, provided that both types of domains recognize essentially the same MHC-peptide antigen. In the specification, “MHC-peptide complex” and “MHC-peptide antigen” have the same meaning. In the context of a peptide that is presented by an MHC molecule, forming an MHC-p complex, the terms “peptide,” “peptidic antigen,” “antigenic epitope” and “antigenic peptide” refer to the same peptide in the complex.

Multivalent TCR domain complexes and therapeutic applications thereof are known in the art. In international patent application publication WO2004/050705, a multivalent TCR domain complex comprising at least two TCR domains, linked by a non-proteinaceous polymer chain or a linker sequence composed of amino acid residues, is disclosed. The disclosed use of the TCR complex is in targeting cell delivery of therapeutic agents, such as cytotoxic drugs, which can be attached to the TCR complex. Di-, tri- and tetravalent TCR complexes are disclosed but divalent TCR complexes are preferred. Importantly, complexes of more than four TCRs are not described. Furthermore, WO2004/050705 focuses solely on the use of a multivalent TCR complex for the delivery of a therapeutic agent, e.g., a toxic moiety for cell killing, to a target cell. It does not teach or suggest the apoptosis-inducing capacity of a multivalent TCR complex itself. The antigen-specific MHC-restricted binding capacity of a multivalent monomeric single-chain protein hereof is sufficient to induce apoptosis of a target cell expressing the relevant antigen. Therefore, using the sole protein hereof only is sufficient for obtaining the desired effect. In, for example, application WO2004/050705, the additive use of an additional or attached cytotoxic agent or toxic moiety is, for example, required.

In the previous application WO2007/073147, it is disclosed that separate individual polypeptide monomers that together build up a multivalent complex of that application, be it antigen-specific MHC-restricted TCRs, TCR-like antibodies or combinations thereof, are post-translationally linked or connected to each other in any suitable manner, be it covalently or non-covalently using standard polypeptide linkage chemistry, in order to achieve the desired pro-apoptotic activity.

Any proteinaceous domain or cluster of domains capable of specifically recognizing and binding to an MHC-peptide complex, comprising either MHC class I or MHC class II proteins, is suitably used in a multivalent apoptosis-inducing single-chain protein. In one embodiment, this protein, comprises at least four, for example, six or even more domains or clusters of domains, connected through regular peptide bonds between the peptide backbone of the domains or clusters of domains building up the multivalent polypeptide, comprising amino acid sequences corresponding to the V_(H) domains of human antibodies.

Exemplified is the generation of a hexavalent mono-specific single-chain monomeric protein, which is specific for a tumor antigen. This hexavalent single-chain protein has therapeutic value in the treatment of cancer. Moreover, the skilled person will appreciate that the disclosure is not limited to any type of antigen, and that hexavalent single-chain proteins are provided that can selectively kill target cells, like, for example, selected aberrant cells, expressing any antigen.

Preferably, the polypeptide is capable of specifically and efficiently recognizing and binding to a cancer-specific epitope or an epitope associated with autoimmune disorders or an epitope presented by any other aberrant cell, for all examples in the context of MHC. Cancer cells may express a group of antigens termed “cancer testis antigens” (CT). These CT are presented as antigenic peptides by MHC molecules (as MHC-p complexes) to CTLs. In fact, these CT are immunogenic in cancer patients as they may elicit anti-cancer responses. They exhibit highly tissue-restricted expression, and are considered promising target molecules for cancer vaccines and other immune intervention strategies.

To date, more than 44 CT gene families have been identified and their expression studied in numerous cancer types. For example, bladder cancer, non-small lung cancer, prostate cancer, melanoma and multiple myeloma express CT genes to a high level. Experiments have shown that expression of these CT genes was indeed testis restricted in healthy individuals. Other antigens that were shown to elicit immune responses in cancer patients include differentiation antigens, such as, for example, the melanoma antigens gp100, Mart-i, Tyrosinase, or antigens that are over-expressed in cancer cells, such as, for example, p53, Her-2/neu, WT-1. Both groups of antigens are not specific for these aberrant cells and are also expressed in healthy tissue, and may therefore elicit autoimmune disease when targeted. In a preferred embodiment, the hexavalent single-chain protein is capable of recognizing and binding to an MHC class I- or to an MHC class II-tumor antigen complex, in particular melanoma associated antigens (MAGE), specifically at tumor cells, leaving healthy cells and tissue essentially unaltered, NB: testis do not present antigens in the context of HLA. The antigen is, for example, a peptide from a member of the CT gene families. The antigen can also be selected from the series of tumor antigens and/or from the series of antigens expressed in the tissue or organ affected by cancer cells, for which it is known that their expression is not tumor specific or not specific for the tissue or organ bearing cancer cells, as is known, for example, for gp100, Mart-1, Tyrosinase, p53, Her-2/neu, WT-1. These antigens are selected as a therapeutic target when the risk for adverse effects is acceptable when related to the beneficial outcome of the treatment with hexavalent single-chain protein, which targets the antigenic peptide complexed with MHC. The general benefit of the disclosure is that, where up until now targets associated with cell surfaces were the predominant goal, intracellular targets now become available through presentation by MHC-1 and/or MHC-2. This means that a renewed survey of intracellular antigens will be carried out to identify intracellular antigens that are tumor specific enough to merit using them as targets in the disclosure. Such a screen has already been carried out in the context of tumor vaccination schemes. Targets that are valuable (because of sufficient specificity, not necessarily efficacy) as tumor vaccine candidates will also be valuable for the disclosure: MAGE-A1, -A2, -A3, -A4, -A5, -A6, -A7, -A8, -A9, -A10, -A11, -A12, -A12, MAGE-B, MAGE-C2, LAGE-1, PRAME, NY-ESO-1, PAGE, SSX-2, SSX-4, GAGE, TAG-1, TAG-2, and HERV-K-MEL.

Human tumor antigens presented by MHC class II molecules have been described, with nearly all of them being associated to multiple myeloma or malignant melanoma. The first antigenic peptide related to a melanoma-specific antigen found was a peptide derived from MAGE-1. Furthermore, three melanoma epitopes were found to originate from the MAGE family of proteins and presented by HLA-DR11 and HLA-DR13. Another set of melanoma antigens, known to contain also MHC class I tumor antigens, comprises Melan-A/MART-1, gp100 and tyrosinase. For an overview of T-cell epitopes that are of use for the disclosure, also see worldwide web at cancerimmunity.org/peptidedatabase/Tcellepitopes.htm.

The first discovered CT, belonging to the group of MAGE-A antigens, has an expression profile that is uniquely restricted to cancer cells and testis cells. However, testis cells are not targeted by the immune system, as they lack expression of MHC molecules. The MAGE-A antigens belong to a family of twelve genes that show high homology. Their expression has been associated with early events in malignant cell transformation and metastatic spread of cancer cells. In addition, down-regulation of MAGE-A expression may induce apoptosis in cancer cells. Within the MAGE-A genes several antigenic epitopes are known by the art. Antigenic peptides usually are presented as 8- or 9-mer amino acid peptides by MHC class I molecules. In addition, antigenic epitopes are known that are present in multiple MAGE-A genes due to the high homology between the different MAGE-A genes. These antigenic epitopes may be considered as multi-MAGE-A epitopes and are presented on cancer cells of various histologic origin. Therefore, they might serve as universal targets for anti-cancer therapy.

MHC molecules are also important as signal-transducing molecules, regulating immune responses. Cross-linking of MHC Class I molecules on B- and T-cells initiates signals that can result in either anergy, or apoptosis, or alternatively in cell proliferation and cytokine production. Several intracellular signaling pathways have been identified that are induced by MHC class I cross-linking. These include 1) phosphorylation of tyrosine kinases, leading to enhanced levels of intracellular calcium ions; 2) activation of the JAK/STAT pathway; and 3) inhibition of PI3K, resulting in the activation of JNK activation. Very high affinity antibodies against MHC that are internalized after binding may induce apoptosis. To be certain in the case of T cell and/or B cell derived tumors, the effect of the molecules may be tested in vitro before initiating therapy.

A further aspect relates to a method for providing the hexavalent single-chain monomeric protein hereof. As described herein above, it typically involves providing a nucleic acid encoding the desired hexavalent polypeptide. This nucleic acid molecule can be introduced, preferably via a plasmid or expression vector, into a prokaryotic host cell and/or in eukaryotic host cell capable of expressing the construct. In one embodiment, a method provides a hexavalent single-chain apoptosis inducing protein comprises the steps of providing a host cell with one or more nucleic acid(s) encoding the hexavalent protein capable of recognizing and binding to a specific MHC-peptide complex, and allowing the expression of the nucleic acids by the host cell.

Preferred host cells are bacteria, like, for example, bacterial strain BL21 or strain SE1, or mammalian host cells, more preferably human host cells. Suitable mammalian host cells include human embryonic kidney (HEK-293) cells or Chinese hamster ovary (CHO) cells, which can be commercially obtained. Insect cells, such as S2 or S9 cells, may also be used using baculovirus or insect cell expression vectors, although they are less suitable when the polypeptides include elements that involve glycosylation. The hexavalent single-chain polypeptides produced can be extracted or isolated from the host cell or, if they are secreted, from the culture medium of the host cell. Thus, in one embodiment, a method comprises providing a host cell with one or more nucleic acid(s) encoding the hexavalent single-chain polypeptide capable of recognizing and binding to a specific MHC-peptide complex, allowing the expression of the nucleic acids by the host cell. Methods for the recombinant expression of (mammalian) proteins in a (mammalian) host cell are well known in the art.

As will be clear, a hexavalent single-chain protein hereof finds its use in many therapeutic applications and non-therapeutic applications, e.g., diagnostics or scientific applications. Provided herein is a method for inducing ex vivo or in vivo apoptosis of a target cell, comprising contacting the cell with a hexavalent single-chain protein hereof in an amount that is effective to induce apoptosis. The target cells can be conveniently contacted with the culture medium of a host cell that is used for the recombinant production of the hexavalent single-chain protein. In one embodiment, it can be used for in vitro apoptosis studies, for instance studies directed at the elucidation of molecular pathways involved in MHC class I and class II induced apoptosis. Hexavalent single-chain proteins hereof may also be used for the detection of (circulating) tumor cells, for the target-cell-specific delivery of cytotoxic compounds or for the delivery of immune-stimulatory molecules.

Preferably, the hexavalent single-chain protein is used for triggering apoptosis of aberrant cells in a subject, more preferably a human subject. For therapeutic applications in humans it is preferred that a hexavalent single-chain protein does not contain amino acid sequences of non-mammalian origin. More preferred are hexavalent single-chain proteins, which only contain human amino acid sequences. Therefore, a therapeutically effective amount of a hexavalent single-chain protein capable of recognizing and binding to a disease-specific epitope can be administered to a patient to stimulate apoptosis of aberrant cells expressing the epitope without affecting the viability of (normal) cells not expressing the disease-specific epitope, e.g., a peptide antigen presented in the context of MHC. It is demonstrated herein that a method hereof allows for the killing of cells in an antigen-specific, MHC-restricted fashion. In a specific embodiment, the disease-specific epitope is a cancer-epitope, for example, a melanoma-specific epitope. The killing of aberrant (tumor) cells while minimizing or even totally avoiding the death of normal cells will generally improve the therapeutic outcome of a patient following administration of the hexavalent single-chain protein.

Also provided is a hexavalent single-chain protein hereof as medicament. In another aspect, provided is the use of a hexavalent single-chain protein for the manufacture of a medicament for the treatment of cancer. For example, a single-chain protein is advantageously used for the manufacture of a medicament for the treatment of melanoma.

Antibody fragments of human origin can be isolated from large antibody repertoires displayed by phages. One aspect hereof is the use of human antibody phage display libraries for the selection of human Fab fragments specific for MHC class I molecules presenting cancer testis antigenic peptides. Antibody Fab fragments specific for MHC class I, HLA-A0201 molecules presenting a multi-MAGE-A epitope have been selected (essentially as described in R. A. Willemsen et al., Cytometry A., 2008, 73:1093-1099) and shown to bind the relevant antigen only. As these antibody-Fab fragments usually display low affinity a method is provided that allows the generation of relatively high avidity antibody chains able to induce apoptosis in a MHC-restricted peptide specific way. An aspect of the disclosure is the development of a single-chain protein molecule comprising multiple antigen binding motifs to enhance MHC-peptide binding avidity, resulting in cross-linking of the MHC-peptide complexes and induction of apoptosis.

An MHC-p complex-specific polypeptide in a multivalent single-chain monomeric protein form hereof is, for example, an MHC-restricted antigen-specific TCR-like antibody (Ab) or functional fragment thereof, which is multimerized at the DNA level in order to obtain a single-chain polypeptide construct upon expression.

Human V_(H) domains usually do not meet the standards for stability and efficient expression that are required by the field. They tend to be unstable and poorly expressed. A process called “camelization” may be used to convert human V_(H) into more stable antibody fragments.

The human antibody germline region V_(H)-3 displays high homology with antibody V_(H) fragments of llamas. Llamas have two types of antibodies, those composed of heavy and light chains, and antibodies that only contain heavy chains. These heavy-chain only antibodies bind antigens similar to classical antibodies composed of heavy and light chains. The smallest functional llama antibody binding domain, the V_(HH) domain, also called single domain antibodies (sdAb), has been shown to be expressed well and may bind antigen with high affinity. In addition, it has been shown that some of the characteristics, such as ease of expression and stability, of llama sdAb can be transferred to, e.g., human V_(H) by replacing a few amino acids in the human V_(H) for those of llama V_(H). High avidity antibody molecules can then be generated by ligation of several “camelized” human V_(H) domains into one single molecule.

Preferred molecules may comprise up to six “camelized” or non-“camelized” human V_(H) domains interspersed by short linkers providing flexibility between the V_(H) domains, thus generating six essentially identical binding domains specific for a single epitope (see, for an example, SEQ ID NO:4 and SEQ ID NO: 13). For example, a hexavalent mono-specific protein is generated that is specific for the HLA-A0201 restricted multi-MAGE-A epitope within a single polypeptide, referred to as a “single-chain protein” or “single-chain polypeptide” or “monomeric protein” or “monomeric polypeptide.” See, for further details, the outlined Examples below. It may be appreciated that this technology allows for the generation of multivalent single-chain proteins that comprise any number of the same or different single domain antibodies. For several reasons (such as, ease of production) repeats are not always the best option. Thus, also contemplated is using different binding domains (essentially recognizing the same target) separated by several different linkers, as shown in FIG. 13.

A hexavalent single-chain monomeric protein hereof, comprising six linearly linked human V_(H) domains is used, for example, to induce apoptosis in cancer cells that express both the MAGE-A genes and HLA-A0201. Noteworthy, specificity for this MHC-peptide complex is provided in this way as cells that do not express HLA-A0201 or that do not express MAGE-A are not killed. See the Examples section for further details. Apoptosis in cancer cells is, for example, detected in vitro by several assays known to the art, including cytotoxicity assays, Tunnel assays and assays detecting active caspases. In animal studies, apoptosis is, for example, revealed by monitoring reduced tumor growth, detection of active caspases or performing a tunnel assay on isolated tumor material.

In literature, it is shown that a single nine amino acid (A.A.) peptide present in MAGE-A2, -A3, -A4, -A6, -A10, and -A12 is presented by HLA-A0201 on tumor cells, and can be recognized by cytotoxic T-lymphocytes.⁽¹⁾ This nine A.A. peptide with sequence Y-L-E-Y-R-Q-V-P-G (SEQ ID NO:7) is almost identical to the HLA-A0201 presented MAGE-A1 peptide Y-L-E-Y-R-Q-V-P-D (SEQ ID NO:9), except for the anchor residue at position 9. Replacement of the anchor residue with Valine results in a 9 A.A. peptide with enhanced binding capacity to HLA-A0201 molecules.⁽¹⁾ Human and mouse T-lymphocytes recognizing the Y-L-E-Y-R-Q-V-P-V (SEQ ID NO:10) peptide presented by HLA-0201 also recognize the original MAGE-A Y-L-E-Y-R-Q-V-P-G (SEQ ID NO:7) and Y-L-E-Y-R-Q-V-P-D (SEQ ID NO:9) peptides presented on tumors of distinct origin. As diverse tumors may each express at least one MAGE-A gene, targeting of this so-called multi-MAGE-A epitope includes the vast majority of tumors. As an example, MAGE-A expression in human prostate tumor cell lines and in human xenographs was analyzed and shown to be highly diverse, but in each individual sample tested at least one MAGE-A gene was expressed (Table 2), confirming that targeting this multi-MAGE-A epitope serves as an essentially universal HLA-A0201 restricted target for therapy.

Of course, several other multi mage or multi target epitopes may be designed. Contemplated are combinations of tumor-specific antigen derived MHC presented epitopes in different HLA restrictions of both MHC-I and MHC-II targeted by multimeric (>=4) binding domains to induce apoptosis in aberrant cell. A number of MHC-peptide combinations that can be targeted (but not limited to) are HLA-A0201/YLEYRQVPG/D (SEQ ID NO:7/9), HLA-CW7/EGDCAPEEK (SEQ ID NO:8), HLA-A24/TFPDLESEK (SEQ ID NO:26) or IMPKAGLLI (SEQ ID NO:27), and HLA-DP4 or HLA-DQ6/KKLLTQHFVQENYLEY (SEQ ID NO:28).

In one embodiment, human antibody fragments specific for the HLA-A0201 presented multi-MAGE-A epitope Y-L-E-Y-R-Q-V-P-V (SEQ ID NO:10) are identified and isolated from a human phage display library. The selected human antibody fragments are optimized regarding their specificity and avidity, and provide the amino acid sequences used for the design and production of hexavalent single-chain polypeptides specific for efficient binding of the HLA-A0201-MAGE-A Y-L-E-Y-R-Q-V-P-G (SEQ ID NO:7) complex, referred to as hexa-AH5. In another embodiment, hexa-AH5 is produced comprising a C-terminal human antibody Fc domain amino acid sequence, providing hexa-AH5Fc with essentially the same or comparable binding characteristics compared to hexa-AH5. In yet another embodiment, hexa-AH5 is produced comprising a C-terminal human serum albumin (HSA) amino acid sequence, providing hexa-AH5HSA with essentially the same or comparable binding characteristics compared to hexa-AH5.

In one embodiment, the hexa-AH5 and/or its equivalents hexa-H5Fc and/or hexa-H5HSA are used in the production of a pharmaceutical composition. In yet another embodiment, hexa-AH5 construct(s) is/are used for the production of a pharmaceutical composition for the treatment of a disease or a health problem related to the presence of aberrant cells exposing the epitope comprising the HLA-A0201-MAGE-A Y-L-E-Y-R-Q-V-P-G (SEQ ID NO:7) complex for hexa-AH5, hexa-AH5Fc and hexa-AH5HSA. The aberrant cells are, for example, tumor cells. In a further embodiment, hexa-AH5 and/or its equivalents hexa-AH5Fc and/or hexa-AH5HSA is used for the treatment of cancer. In yet another embodiment, hexa-AH5 and/or its equivalent, is used, for example, for the treatment of prostate cancer, breast cancer, multiple myelomas or melanomas.

The disclosure is exemplified by the Examples below.

ABBREVIATIONS USED

A.A., amino acid; Ab, antibody; ADA, anti-drug antibodies; AFP, alpha-fetoprotein; APC, antigen presenting cell; β2-M, β2-microglobulin; CDR, complementarity determining region; CEA, carcino-embryonic antigen; CHO, Chinese hamster ovary; CT, cancer testis antigens; CTL, cytotoxic T-lymphocyte; DC, dendritic cell; EBV, Epstein-Barr virus; ELISA, enzyme linked immunosorbent assay; HEK, human embryonic kidney; HLA, human leukocyte antigen; i.v., intravenously; kDa, kilo Dalton; MAGE, melanoma-associated antigen; MHC, major histocompatibility complex; MHC-p, MHC-peptide; PBSM, PBS containing 2% non-fat dry milk; sc-Fv, single-chain variable fragment; V_(HH) or sdAb, single domain antibodies; TCR, T-cell receptor; VH, Vh or V_(H), variable amino acid sequence of an antibody heavy domain.

EXAMPLES Example 1: Selection of Human Antibody Fragments Specific for HLA-A0201/Multi-MAGE-A

1.1

To obtain human antibody fragments specific for the HLA-A0201 presented multi-MAGE-A epitope Y-L-E-Y-R-Q-V-P-G (SEQ ID NO:5) a Human Fab phage display library was constructed according to the procedure previously described by de Haard et al.⁽²⁾ and used for selections essentially as described by Chames et al.⁽³⁾ Human Fab phages (10¹³ colony-forming units) were first pre-incubated for 1 hour at room temperature in PBS containing 2% non-fat dry milk (PBSM). In parallel, 200 μl Streptavidin-coated beads (Dynal) were equilibrated for 1 hour in PBSM. For subsequent rounds, 100 μl beads were used. To deplete for pan-MHC binders, each selection round, 200 nM of biotinylated MHC class I-peptide (MHC-p) complexes containing an irrelevant peptide (Sanquin, the Netherlands) were added to the phages and incubated for 30 minutes under rotation. Equilibrated beads were added, and the mixture was incubated for 15 minutes under rotation. Beads were drawn to the side of the tube using magnetic force. To the depleted phage fraction, subsequently decreasing amounts of biotinylated MHC-p complexes (200 nM for the first round, and 20 nM for the second and third round) were added and incubated for 1 hour at room temperature, with continuous rotation. Simultaneously, a pan-MHC class I binding soluble Fab (D3) was added to the phage-MHC-p complex mixture (50, 10, and 5 μg for rounds 1-3, respectively). Equilibrated streptavidin-coated beads were added, and the mixture was incubated for 15 minutes under rotation. Phages were selected by magnetic force. Non-bound phages were removed by five washing steps with PBSM, five steps with PBS containing 0.1% TWEEN®, and five steps with PBS. Phages were eluted from the beads by 10 minutes incubation with 500 μl freshly prepared tri-ethylamine (100 mM). The pH of the solution was neutralized by the addition of 500 μl 1 M Tris (pH 7.5). The eluted phages were incubated with logarithmic growing E. Coli TG1 cells (OD_(600nm) of 0.5) for 30 minutes at 37° C. Bacteria were grown overnight on 2×TYAG plates. The next day, colonies were harvested, and a 10 μl inoculum was used in 50 ml 2×TYAG. Cells were grown until an OD_(600m) of 0.5, and 5 ml of this suspension was infected with M13k07 helper phage (5×10¹¹ colony-forming units). After 30 minutes incubation at 37° C., the cells were centrifuged, resuspended in 25 ml 2×TYAK, and grown overnight at 30° C. Phages were collected from the culture supernatant, as described previously, and were used for the next round panning. After three selection rounds a 261-fold enrichment was obtained, and 46 out of 282 analyzed clones were shown to be specific for the HLA-A2-multi-MAGE-A complex (FIG. 1). ELISA using the HLA-A0201/multi-MAGE-A complexes as well as HLA-A0201 complexes with a peptide derived from JC virus was used to determine the specificity of the selected Fab.

1.2 Human Fab Specific for the HLA-A0201/Multi-MAGE-A Epitope Bind Antigen-Positive Cells

Selected Fab phages were then analyzed for their capacity to bind HLA-A0201-positive EBV-transformed B-LCL loaded with the multi-MAGE-A peptide Y-L-E-Y-R-Q-V-P-V (SEQ ID NO:10). The B-LCL line BSM (0.5×10⁶) was loaded with multi-MAGE-A peptide (10 μg in 100 ptl PBS) for 30 minutes at 37° C., followed by incubation with the Fab phages AH5, CB1, CG1, BD5 and BC7 and analyzed by flow-cytometry. As shown in FIG. 2, Fab AH5, CB1 and CG1, specifically bound to the peptide loaded cells only, whereas Fab BD5 and BC7 displayed non-specific binding to BSM that was not loaded with the multi-MAGE-A peptide. No binding was observed by AH5, CB1 and CG1 to non-peptide loaded cells.

Phages presenting AH5, CB1 and CG1, as well as the HLA-A0101/MAGE-A1-specific Fab phage G8⁽⁴⁾ were then used to stain tumor cell lines of distinct histologic origin. To this end prostate cancer cells (LNCaP), multiple myeloma cells (MDN), melanoma cells (MZ2-MEL43 and G43), and breast cancer cells (MDA-MB157) were stained and analyzed by flow cytometry (FIG. 3). The Fab AH5 specifically bound multiple myeloma cells MDN, and not the HLA-A0201-negative melanoma and breast cancer cells. Both CB1 and CG1 displayed non-specific binding on the melanoma cell line G43. The positive control Fab G8 demonstrated binding to all cell lines tested.

1.3 Fab AH5 Binds HLA-A0201/Multi-MAGE-A Complexes Only

ELISA using multiple peptide/MHC complexes then confirmed the specificity of Fab-AH5. To this end HLA-A0201 complexes presenting peptides multi-MAGE-A, gp100, JCV and MAGE-C2, as well as a HLA-A1/MAGE-A1 complex were immobilized on 96-well plates and incubated with phages displaying Fab AH5 and control Fab G8. As shown in FIG. 4, AH5 only binds HLA-A0201/multi-MAGE-A and not the irrelevant complexes HLA-A0201/gp100, HLA-A0201/MAGE-C2, HLA-A0201/JCV and HLA-A0101/MAGE-A1. The positive control Fab G8 only binds to its relevant target HLA-A0101/MAGE-A1.

Example 2: Production of Hexameric Proteins Comprising Camelized Single Domains AH5 VH Domains 2.1 Design of Genes for Production of Hexameric AH5 VH Proteins

Human antibody germline gene VH3 demonstrates high homology to llama single domains VHH. Exchange of amino acids 44, 45 and 47 in the human VH3 genes by amino acids present in llama VHH at these positions has shown to enhance stability and expression of the human VH3 genes.⁽⁵⁾ The AH5 VH demonstrates a low homology to germline gene VH3-33*01 (71% as determined by IMGT homology search) however, its expression and stability might benefit from the exchange of amino acids 44, 45 and 47 by llama VHH amino acids, a process called camelization. In addition a gene was compiled that upon expression would comprise six AH5 VH domains. To this end, a gene called hexa-AH5 was designed comprising the pelB secretion signal, which was operatively linked to six codon-optimized, camelized AH5 VH domains with GSTSGS linkers between each AH5 VH domain (see hexa-AH5, see SEQ ID NO:1 for the DNA sequence and SEQ ID NO:4 for the amino acid sequence). This gene was synthesized by “Geneart” (Regensburg, Germany) and cloned into the pStaby 1.2 vector (Delphi genetics, Belgium) for expression in E. coli.

2.2 Production and Purification of Hexameric AH5 VH Protein

For expression of hexameric AH5 VH proteins (hexa-AH5, see SEQ ID NO: 1 for the DNA sequence and SEQ ID NO:4 for the amino acid sequence) the pStaby-Hexa-AH5 vectors were introduced via electroporation into SE1 bacteria. Positive clones were grown in the presence of 2% glucose at 30° C. until OD₆₀₀=0.8. Bacterial TYAG medium was then replaced with TY medium containing 1 mM IPTG to induce expression. After overnight culture at 30° C. bacteria and medium were harvested. The periplasm fraction was collected after incubation of bacteria with PBS/EDTA/NaCl for 30 minutes on ice. Protein expression was then analyzed by SDS-PAGE. As shown in FIG. 5, Hexa-AH5 protein was secreted into the medium and was present in the bacterial periplasm.

Hexameric AH5 VH proteins were isolated from media and bacteria using Ni-affinity purification. To this end, medium was incubated with Ni-coupled Sepharose-beads and incubated overnight, while stirring gently. To obtain intracellular proteins bacteria were lysed and cellular debris removed by centrifugation. After overnight dialysis with PBS Hexameric AH5 VH proteins were purified with Ni-Sepharose. Purity of the Hexameric AH5 VH proteins was checked by SDS-PAGE and protein concentration determined by BCA protein assay (Pierce).

Example 3: Hexameric AH5 VH Proteins Induce Apoptosis in Diverse Tumor Cells

Cross-linking of MHC class I molecules by pan-MHC class-I and 32M-specific antibodies results in the induction of apoptosis.⁽⁶⁾ This process was shown to be caspase-9 dependent and results in the eradication of MHC class I-positive tumor cells in vitro and in vivo. The induction of apoptosis by pan-MHC class I antibodies and anti-β2M-specific antibodies is not specific for tumors expressing tumor-specific antigens. In contrast, cross-linking of peptide/MHC molecules through the interaction of molecules that resemble T-cell receptors binding to specific peptide/MHC complexes will result in tumor-specific apoptosis induction. Efficient cross-linking will depend on the number of peptide/MHC complexes that are simultaneously bound by the therapeutic molecule.

3.1 Hexameric AH5 Protein Kills Diverse Tumor Cells

The hexameric AH5-VH proteins were analyzed for their capacity to induce apoptosis by incubation with diverse tumor cells, known to express both HLA-A0201 and MAGE-A genes. The cell-lines Daju, Mel 624 (melanoma), PC346C (prostate cancer), as well as MAGE-A-negative cells (911 and HEK293T) were incubated with 10 μg/ml Hexa-AH5 protein (in DMEM medium, supplemented with pen/strep, Glutamine and non-essential amino acids). Four hours later, cells were visually inspected for classical signs of apoptosis, such as detachment of the cells from tissue culture plates and membrane blebbing. As shown in FIG. 6, Daju cells indeed detach from the tissue culture plates only after incubation with the Hexa-AH5 protein. This was also seen for the Mel 624 and PC346C cells. When incubation was extended to overnight, Daju, Mel624 and PC346C cells were disintegrated and notably absent in the treated cultures. Cells that were not treated with the hexa-AH5 protein were not affected, as well as cells that do not express HLA-A0201 (HEK293T) and MAGE-A genes (911 and HEK293T).

3.2 Hexameric AH5 Protein Induces Active Caspase-3

A classical intra-cellular hallmark for apoptosis is the presence of active caspase-3. To determine whether or not the Hexameric AH5 proteins induce active caspase-3, Daju cells were incubated with 10 μg/ml Hexa-AH5 protein. After four hours FAM-DEVD-FMK (SEQ ID NO:25), a fluorescently labeled inhibitor for caspase-3/7 was added to the tissue culture medium. This substrate can pass the cell-membrane and only when active caspase-3 is present, a bright fluorescent signal will be detected by, e.g., fluorescent microscopy.

As shown in FIG. 7, Daju cells treated with Hexa-AH5 protein are emitting a fluorescence signal demonstrating the presence of active caspase-3. Cells that were not treated did not show fluorescence, demonstrating the specificity of the caspase-3 inhibitor.

Example 4: Hexameric AH5 Protein Induces Apoptosis in a Transplantable Human Tumor Model

To demonstrate apoptotic activity of the Hexa-AH5 proteins in three-dimensional human tumors, an orthotopic prostate cancer model was used. To this end, human PC346C prostate cancer cells were injected into the prostate of male NOD-SCID mice and allowed to grow until tumors were detectable by ultrasound guided inspection.

4.1 Intra-Tumoral Injection of Hexa-AH5 Results Induces Apoptosis

The human PC346C prostate tumors in NOD-SCID mice were injected once directly with 10 μg Hexa-AH5 protein (in 20 μl total volume). The next day, tumors were removed, fixed and paraffin embedded. Slides were prepared from the paraffin-embedded tumors and stained with the Tunnel Universal Apoptosis Detection Kit (Genescript), an assay that detects fragmented DNA, a classical marker of apoptosis. In brief, slides were heated for 30 minutes at 60° C., washed three times with PBS, and incubated for one hour with proteinase K solution. Slides were then incubated with blocking solution (3% H₂O₂ in methanol) for 10 minutes, washed with PBS and incubated for one hour at 37° C. with Tunnel reaction mixture (equilibrium buffer, Biotin-11-dUTP, and TdT). After three washes slides were incubated with Streptavidin-HRP solution for 30 minutes at 37° C., and finally incubated with DAB-substrate (DAB-buffer, H₂O₂ in PBS).

Microscopic analysis of tumor material treated with Hexa-AH5 demonstrates large areas of apoptotic cells (see FIG. 8). Untreated tumors do not show any signs of DNA damage

4.2 Intravenous Injection of Hexa-AH5 Induces Apoptosis in Orthotopic Prostate Cancer Cells

4.2.1 Prostate Tumor Cells Demonstrate Nicked DNA after i.v. Injection with Hexa-AH5

In a next experiment NOD-scid mice with the orthotopic human PC346C prostate tumor were injected once via tail vain with 25 μg Hexa-AH5 (in 150 jil total volume). The next day, tumors were removed, paraffin embedded and tumor slides stained for Nicked DNA with the Tunnel assay.

As shown in FIG. 9, large areas of apoptotic cells are present in Hexa-AH5-treated mice, whereas non-treated mice did not show any signs of apoptosis.

4.2.2 Prostate Tumor Cells Demonstrate Active Caspase after i.v. Injection with Hexa-AH5

NOD-scid mice with the orthotopic PC346C tumor were injected once via tail vain with 25 μg Hexa-AH5 (in 150 μl total volume). The next day, these mice received an injection with FLIVO (Immunohistochemistry Ltd.), a fluorescently labeled caspase inhibitor. This inhibitor was allowed to circulate and pass cellular membranes for one hour. Tumors were then removed, fixed and paraffin embedded.

Analysis of Hexa-AH5-treated tumors by fluorescence microscopy demonstrated the presence of numerous cells that stained positive for the caspase substrate (see FIG. 10). No fluorescently labeled cells were detected in untreated mice.

Example 5: Construction of Hexa-AH5 Genes to Improve Circulation and Tumor Penetration

The pharmacokinetic properties of therapeutic proteins, e.g., their distribution, metabolism and excretion are dependent on factors, such as shape, charge and size. Most small plasma molecules (MW<50-60 kDa) possess very short half-life, whereas larger plasma proteins, such as human serum albumin (HSA) and immunoglobulins (Ig) have very long half-lives (19 days for HSA, 1-4 weeks for Ig). Indeed, addition of IgG-Fc or Human serum albumin has shown to extend circulation time, tumor penetration and antitumor effects when linked to therapeutic proteins.

5.1 Construction of Hexameric AH5 with IgG1-Fc and Human Serum Albumin

The Hexameric AH5 construct was linked to the IgG1-Fc region or to human serum albumin, codon optimized for expression in eukaryotic cells and cloned into the pcDNA-3.1+ vector (Geneart, Regensburg, Germany) (see DNA sequence with SEQ ID NO:2 and amino acid sequence with SEQ ID NO:5 for hexa-AH5Fc, and see DNA sequence with SEQ ID NO:3 and amino acid sequence with SEQ ID NO:6 for hexa-AH5HSA, respectively).

5.2 Hexameric AH5-Fc and AH5-HSA Induce Active Caspase-3

The hexameric AH5-FC and AH5-HSA constructs, cloned into pcDNA-3.1+, were expressed in 293T cells. Supernatant obtained four days after transfection was used to induce apoptosis in melanoma 624 cells known to express HLA-A0201 and MAGE-A genes. To this end, melanoma 624 cells were seeded in 24-well plates (0.25×10⁶ cells/well) and allowed to attach overnight. The next day, medium was replaced with medium obtained from transfected 293T cells. Results showed positive caspase-3 staining for 624 melanoma cells treated with both hexa-AH5-Fc and Hexa-AH5-HSA. No staining was observed for 624 cells incubated with plain medium or HLA-A0201 positive, MAGE-A-negative 911 cells (FIGS. 11A and 11B).

5.3 Extended Survival of Mice and Delayed Tumor Growth of Mice Treated with Hexameric AH5

Mice inoculated with melanoma cell line Daju (HLA-A0201/MAGE-A positive) were treated with intravenous injections of hexameric AH5 protein (2.5 ug/2 times/week). Shown are 1) tumor free mice, and 2) tumor growth (FIG. 12).

5.4 Enhanced Induction of Apoptosis by Dimeric Hexameric AH5CH1 and 11HCH1.

For expression in eukaryotic cells the AH5CH1 and 11HCH1 sequences were introduced into the pMSec SUMOSTAR vector (Hexameric AH5CH1 and 1lHCH1 were produced in supernatant of 293T cells after transfection with CaPO4. One hour after incubation of Daju and MEL624 melanoma cells with 293T supernatant (1:1 diluted in DMEM, 5% FCS) membrane blebbing and detachment of cells were observed.

5.5 Improved Expression of Hexameric AH5 at 25° C.

Expression of Hexameric AH5 in SE1 at 30° C. or 37° C. in shaking flasks was shown to result in many unwanted smaller products. Lowering the temperature during growth and production to 25° C. resulted in a marked improvement of production. Less, to no side products were obtained as well as a higher yield of the protein (FIGS. 14A and 14B).

TABLE 1 Examples for the frequency of MAGE-A expression by human cancers Table 1: Examples for the frequency of MAGE-A expression by human cancers Frequency of expression (%) MAGE- MAGE- MAGE- MAGE- MAGE- MAGE- MAGE- cancer A1 A2 A3 A4 A6 A10 A11 Melanoma 16 E 36 E 64 E 74 Head and neck 25 42 33  8 N N N Bladder 21 30 35 33 15 N  9 Breast  6 19 10 13  5 N N Colorectal N  5  5 N  5 N N Lung 21 30 46 11  8 N N Gastric 30 22 57 N N N N Ovarian 55 32 20 E 20 N N osteosarcoma 62 75 62 12 62 N N hepatocarcinoma 68 30 68 N 30 30 30 Renal cell 22 16 76 30 N N N carcinoma E, expressed but the frequency is not known; N, expression by tumors has never been determined or observed

TABLE 1B Expression analysis of MAGE-A1-A6 genes detected by nested RT-PCR with common primers in squamous cell carcinoma of the head and neck. Primary site % of positive expression Larynx 72.7% (8/11)  Hypopharynx 100% (2/2)  Base of tongue 50% (1/2) Tonsil 100% (2/2)  Total (n = 17)  76.5% (13/17) Adapted from: ANTICANCER RESEARCH 26: 1513-1518 (2006)

TABLE 2 MAGE-A expression in human prostate cancer cell lines and prostate cancer xenografts. Table 2: MAGE-A expression in human prostate cancer cell lines and prostate cancer xenografts. Cellline/ MAGE- Xenograft A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 LNCaP + ++ ++ ++ + PC346C + ++ ++ + ++ + + ++ OVCAR + + + + JON ++ ++ ++ + + PNT 2 + + + + + C2 SD48 + + + + PC-3 + + + PC 374 + PC 346p + ++ ++ ++ + ++ + PC 82 + + PC 133 ++ + + PC 135 + PC 295 + PC 324 + + + PC 310 + ++ + ++ + PC 339 ++ ++ + ++ + + + Expression of the MAGE-A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11 and A12 genes in diverse prostate tumor cell lines and prostate xenografts was analyzed by RT-PCR. Shown are expression levels in individual samples tested. Blank = no expression, + = low expression, ++ = high expression. All cell lines/xenografts express at least one MAGE-A gene.

REFERENCES

-   (1) Stephanie Graff-Dubois, Olivier Faure, David-Alexandre Gross,     Pedro Alves, Antonio Scardino, Salem Chouaib, Francois A. Lemonnier     and Kostas Kosmatopoulos. Generation of CTL Recognizing an     HLA-A*0201-Restricted Epitope Shared by MAGE-A1, -A2, -A3, -A4, -A6,     -A10, and -A12 Tumor Antigens: Implication in a Broad-Spectrum Tumor     Immunotherapy. The Journal of Immunology, 2002, 169:575-580. -   (2) Hans J. de Haard, Nicole van Neer, Anneke Reurs, Simon E.     Hufton, Rob C. Roovers, Paula Henderikx, Adriaan P. de Bruine,     Jan-Willem Arends, and Hennie R. Hoogenboom. A Large Non-immunized     Human Fab Fragment Phage Library That Permits Rapid Isolation and     Kinetic Analysis of High Affinity Antibodies. The Journal of     Biological Chemistry, 1999, 274:18218-18230. -   (3) P. Chames, H. R. Hoogenboom, and P. Henderikx. Selection of     antigens against biotinylated antigens. In Antibody phage display,     methods and protocols, Edited by P. M. O'Brien and R. Aitken.     Methods in Molecular Biology 2002, 178:147-159. -   (4) Patrick Chames, Simon E. Hufton, Pierre G. Coulie, Barbara     Uchanska-Ziegler, Hennie R. Hoogenboom. Direct selection of a human     antibody fragment directed against the tumor T-cell epitope     HLA-A1-MAGE-A1 from a nonimmunized phage-Fab library. PNAS, 2000.     97:7969-7974. -   (5) Lutz Riechmann, Serge Muyldermans. Single domain antibodies:     comparison of camel VH and camelized human VH domains. Journal of     Immunological Methods 1999, 231:25-38. -   (6) Jing Yang, PhD and Qing Yi. Killing Tumor Cells Through Their     Surface b2-Microglobulin or Major Histocompatibility Complex Class I     Molecules. Cancer 2010. 116:1638-1645. 

1.-27. (canceled)
 28. A single polypeptide chain comprising: at least four specific binding domains, the specific binding domains separated by linker amino acid sequences, wherein each specific binding domains comprises an immunoglobulin fragment.
 29. The single polypeptide chain of claim 28, further comprising: a peptide that is not a linker and does not comprise an immunoglobulin fragment.
 30. The single polypeptide chain of claim 28, having six specific binding domains.
 31. The single polypeptide chain of claim 28, wherein the at least one specific binding domain is a V_(H).
 32. The single polypeptide chain of claim 28, wherein the specific binding domains are able to bind to an MHC-I-peptide complex.
 33. The single polypeptide chain of claim 32, wherein the MHC-I-peptide complex comprises a peptide derived from a tumor related antigen.
 34. The single polypeptide chain of claim 31, wherein at least one of the specific binding domains comprises SEQ ID NO:11 or SEQ ID NO:12.
 35. The single polypeptide chain of claim 31, wherein at least one of the linker amino acid sequences comprises SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24.
 36. The single polypeptide chain of claim 31, wherein the specific binding domains are able to bind to the MHC-peptide complex, but not to the peptide itself, another MHC-peptide complex, or an empty MHC.
 37. A method for producing the single polypeptide chain of claim 28, the method comprising: culturing a host cell comprising a polynucleotide encoding the polypeptide, allowing for expression of the polynucleotide, and harvesting the polypeptide.
 38. A pharmaceutical composition comprising: the single polypeptide chain of claim 28, and a suitable diluent and/or excipient.
 39. The pharmaceutical composition according to claim 38, further comprising a cytostatic and/or tumoricidal agent.
 40. A conjugate of the single polypeptide chain of claim 28, and a cytostatic or tumoricidal agent.
 41. A single polypeptide chain consisting of: six specific binding domains, the specific binding domains separated by linker amino acid sequences, wherein each specific binding domains comprises an immunoglobulin fragment; and a peptide that is not a linker and does not comprise an immunoglobulin fragment.
 42. The single polypeptide chain of claim 41, wherein the specific binding domains are each V_(H).
 43. A single polypeptide chain consisting of: six specific binding domains, the specific binding domains separated by linker amino acid sequences, wherein each specific binding domains comprises a V_(H); and a peptide that is not a linker and does not comprise a V_(H). 